Telomeric DNA is packed into a protective complex that acts as a cap over the chromosome end. This cap is essential for preventing chromosome fusions and hence the chromosomal rearrangements that result in cancer. The telomeric cap is a dynamic structure that balances the need to protect the DNA terminus from the DNA repair machinery with the need to allow access to telomerase and other replication enzymes. The architecture of the cap is still unclear, but it seems to be composed of a number of molecular interactions that include DNA-protein or protein-protein interactions with the single-strand overhang on the DNA terminus, the telomeric tract, and subtelomeric sequences. This proposal focuses on the single-strand overhang and its associated proteins because it is a particularly critical component of the cap. The proposal has two main goals: to define the architecture of the telomeric cap by ascertaining how factors that associate with or modulate overhang structure promote cap formation, and to determine how the DNA terminus is processed to generate the precise overhang structure that is required to form a functional cap. The specific aims are as follows: 1. To characterize the telomeric DNA structure generated by leading-strand synthesis in Tetrahymena. This aim tests current models for telomere replication and will establish the structures from which G-overhangs are generated. 2. To investigate the role of telomerase and repair proteins in telomere capping and G-overhang generation and maintenance. This will be achieved by deleting or mutating TERT, Ku70 or Rad51 and determining the effect on overhang structure and cap architecture. 3. To delineate the role of the Tetrahymena G-overhang binding protein in telomere capping and determine whether this protein specifies the boundaries for overhang processing. The Tetrahymena Pot1 homolog (tPot1) will be identified and the effect of tPot1 deletion or mutagenesis on capping and overhang processing will be determined. 4. To characterize the nucleases responsible for G- and C-strand cleavage. In vivo and in vitro processing assays will be developed with artificial telomere substrates. The proposed studies will give a much deeper understanding of the DNA processing reactions that lead to assembly of the terminal DNA-protein complex, the architecture of this telomeric cap, and its role in chromosome protection and stabilization.